Cancer is the second leading cause of death in the United States-second only to diseases of the heart-and accounts for about 23% of all deaths. Although significant progress has been made in the treatment of many cancers, it is estimated that in 2010, 569,490 Americans will die from these diseases (Cancer Facts and Figures 2010, American Cancer Society) and there is as yet no efficacious treatment for many forms of cancers. The molecular complexity of cancer makes the identification of additional oncogenic pathways amenable to therapeutic intervention paramount. In June 2010, NIH posted a program announcement (PA-10- 213) for the Development of Assay for High-throughput Screening for Use in Probe and Pre-therapeutic Discovery to encourage the development of novel, scientifically outstanding assays that have the potential to be developed into high-throughput screens (HTS) of relevant, high priority disease targets both for the identification of small molecule tools and therapeutic candidates. Together, we and our collaborators present data that provide compelling evidence that the inhibition of cytohesins, the guanine nucleotide exchange factors that activate Arf family GTPases, produces anticancer activity in vastly different cell lineages. Further, we demonstrate that this inhibition reduces in vitro proliferation and invasion in the context of Ras/Raf/ERK constitutive activation. Mutations of these downstream components of receptor tyrosine kinase signaling pathways have greatly limited the utility and effectiveness of currently available cancer therapies. Therefore, we believe targeting the cytohesin, ARNO, and its effector, Arf6, may produce promising new small molecule inhibitors useful at combating both innate and acquired chemotherapeutic resistance. In this application we seek to specifically address the need defined by NIH and present a strategy to developing assays amenable to a HTS campaign intended for the identification and evaluation of ARNO and/or Arf6 negative modulators. If successful, we will then seek to automate these screens through collaborations with the NIH's recently launched Molecular Libraries and Imaging initiative or other institutions with access to large, diverse compound libraries. In the long term, this strategy promises to identify new small molecule probe and pre-therapeutic compounds against these novel oncogenic targets.